DESCRIPTION: The long-term objective of this research is to use vibrational probes and infrared (IR) spectroscopy to study protein hydration and structure. The proposed azides are sensitive, site- specific, and non-perturbative vibrational probes with sufficient spatial and temporal resolution to study the molecular dynamics of interest. The azide probes will be used to investigate hydration dynamics in three proteins: adenylate kinase (AK), superfolder green fluorescent protein (sfGFP), and triosephosphate isomerase (TIM). Mutations in AK are associated with hemolytic anemia, reticular dysgenesis, and ciliary dyskinesia. TIM deficiency results in chronic hemolytic anaemia, cardiomyopathy, susceptibility to infections, neurological dysfunction, and decreased life spans in humans and TIM from the parasite Trypanosoma brucei has been identified as the causative agent of sleeping sickness. The proposed research involves the synthesis of adenosine azides 1a-1f and phenylalanine azides 2a-2e. The ability of azides 1 to serve as effective IR probes of hydration and electrostatics in AK will be investigated by linear IR and then sent to a collaborator, Matthew Tucker, for 2D IR analysis to measure hydration dynamics around the azide probe. Azides 2 have rigid linkers that allow the distance between the azide and the residue side chain to be varied in a controlled and systematic fashion. Azides 2 will be synthesized and genetically incorporated into sfGFP and TIM where they will serve as molecular hydration rulers. Linear IR spectroscopy will be used to investigate the hydration and electrostatics of these proteins and a collaborator, Christopher Cheatum, will perform 2D IR experiments to measure hydration dynamics around the azide probe. The bioorthogonal reactivity of azides will be used to ligate azide 2b at the 150 site in sfGFP by a click reaction with an alkyne metal carbonyl to directly compare azides and metal carbonyls as vibrational hydration probes. The ability to extract the distance and angle between two azide isotopomers from the strength of anharmonic coupling between them will be investigated with 2D IR spectroscopy using azides 1 bound to AK and azides 2 incorporated into AK. Collaborator Steven Corcelli will use computational methods to model hydration dynamics and will aid in the development of a model to describe the distance dependence of anaharmonic coupling between azido groups. Another goal of this health related research is to train a minimum of six undergraduate students in synthetic, biochemical, and spectroscopic techniques.